ABSTRACT BK and JC Polyomaviruses cause significant morbidity and mortality in immune suppressed patients, including progressive multifocal leukoencephalopathy (PML), polyomavirus associated nephropathy (PVAN), and hemorrhagic cystitis. Currently there is no effective therapy, prophylaxis, or vaccination for any polyomavirus. In addition, BK/JC viruses replicate in the renal tubular and urothelial epithelium, so that infection is maintained in the ?immune privileged? urinary space. Hence, there is an unmet need for a potent therapeutic/prophylactic capable of targeting BK/JC viruses in the urinary space. The overall goal of the proposed project is to develop a novel multivalent antibody-based therapy for BK and JC polyomavirus infections. In the proposed work, TeneoBio, Inc. will utilize a platform based on the production of fully human heavy chain antibodies in genetically engineered rats (UniRat system). Unlike conventional antibodies, heavy chain antibodies can be engineered to consist of only two heavy chains (without light chains), an isolated heavy chain, or a ?string? of variable domains (so called ?domain UniAbs? or UniDabs). Due to their small size, UniDAbs can be filtered into the urine, thus targeting BK/JC viruses that reside in the urinary space. In addition, since UniDAbs are fully human, adverse side effects associated with humanized, murine, and alternative multivalent antibody approaches, such as anti-drug antibodies, are not anticipated. It has been shown that HCAs/UniDAbs as no more immunogenic than conventional Abs. In this proposal we first plan to generate heavy chain antibodies from UniRats immunized with BK/JC-derived virus like particles by developing an optimal immunization protocol for BK/JC virus antibodies in UniRats, followed by an identification of the resulting BK/JC virus specific heavy chain antibody leads (approximately 300) using next generation sequencing. Next, we will implement a 2-stage screening approach to select 5-10 high affinity anti-BK/JC heavy chain antibodies. In the first stage we will search for sequence families that demonstrate high affinity binding and neutralization of multiple BK/JC serotypes; In the second stage, an additional round of high throughput vector assembly, expression, and neutralization assays will be performed to yield 5-10 stable antibody candidates. Finally, the antibodies with the highest affinity will be further characterized by screening for broad neutralization of additional BK/JC variants. The selected broadly neutralizing heavy chain antibodies/UniDAbs will undergo functional characterization to aid the generation of stable UniDab strings in a future phase 2 of this project. Upon successful completion of the proposed study at least 1-3 heavy chain antibodies will be selected. These antibodies will serve in phase II as the basis for construction of high affinity virus-specific UniDabs. These UniDabs will then be tested in pre-clinical and finally clinical applications.